Field of the Invention
The invention relates to a vector and its use for the expression of therapeutically relevant genes in mammal cells. Fields of application of the invention are in the biotechnology industry, the pharmaceutical industry, and in medicine.
In the treatment of cancer patients with cytostatic agents, signs of resistance frequently occur (Igor B. Roninson, 1991), which are an obstacle to achieving the desired therapeutic result. For this reason, a plurality of different chemotherapeutic regimens have been developed, varying the application of the cytostatic agents and their dosage. In addition, attempts were made to enhance chemotherapy by combining cytostatic agents with other groups of substances, such as, e.g., cytokines, monoclonal antibodies, etc. Another way of rendering tumor chemotherapy more effective is the use of antibody-coupled cytostatic agents in order to thereby achieve the transport of the cytostatic agent to the cancer cell and to attain effectiveness in this cell (H. M. Pinedo, et al. 1991). Although partial success has been achieved in some cases, a thorough clinical success could as yet not be achieved with any of these methods.
Successful chemotherapy of human tumors is often limited by simultaneous resistance to structurally and functionally unrelated amphiphilic cytotoxic drugs, the multidrug resistance (MDR) phenotype (Germann, et al, 1993). Classical MDR is mediated by the multidrug resistance gene (mdr1) encoded P-glycoprotein, which acts as an energy-dependent drug-efflux pump (Roninson, 1992). Several studies have demonstrated the inducibility of the mdr1 gene promoter by stress factors, such as differentiating agents, heat shock, arsenite, protein kinase c agonists, as well as by UV irradiation (Mickley et al, 1989; Chin et al, 1990; Chaudhary et al, 1992; Uchiumi et al, 1993; Ferrandis et al, 1993). Modulating effects of MDR associated drugs on the mdr1 promoter have also been reported (Kohno et al, 1989; Licht et al, 1991; Chaudhary et al, 1993; Stein et al, 1994), mostly using the COS monkey kidney system. However, since colon carcinoma cells frequently show high intrinsic mdr1 expression levels, they may represent an important model for investigation of modulated mdr1 expression and the resulting MDR phenotype.
Among human sarcomas, osteosarcomas usually display high intrinsic mdr1 expression while malignant fibrous histiocytomas (MFH) do not. A comparative polymerase chain reaction (PCR)-based sequence analysis of the mdr1 promoter revealed point mutations in 7/9 osteosarcomas at nucleotides +103 (2 cases T.fwdarw.C) and +137 (5 cases G.fwdarw.T). No changes were seen in 8 MFHs. When COS cells transfected with CAT constructs containing the T.fwdarw.C-mutant mdr1 promoters were treated with vincristine or doxorubicin, expression of the chloramphenicol acetyltransferase (CAT) gene was enhanced to a higher extent than with constructs containing wild-type or G.fwdarw.T-mutant mdr1 promoters.
Several investigations have shown that malignant gliomas express P-glycoprotein at high levels. The P-glycoprotein is a product of the multidrug resistance gene (mdr1) and functions as an energy-dependent efflux pump which decreases drug accumulation and cytotoxicity. Since tumor necrosis factor alpha (TNF.alpha.) is a powerful anticancer agent used in clinical trials and gene therapy protocols, this cytokine gene was chosen for the present investigations. Transduction of the human TNF.alpha.(hTNF) gene carrying retroviral vector pN2tk-hTNF into U373MG human glioblastoma cells resulted in expression and secretion of biologically active hTNF. Release of transduced hTNF reduces P-glycoprotein expression and is associated with enhanced rhodamine-123 uptake and potentiation of cytotoxicity of the MDR relevant drugs vincristine and doxorubicin. Furthermore the transfected cell clones showed a reduced growth rate compared to the parental cells.
Malignancies of the brain including gliomas have poor prognosis due to their aggressiveness and their resistance to radiotherapy and chemotherapy. Several investigations have been made to identify the molecular mechanisms of intrinsic resistance in human and animal brain tumors and tumor models (Mousseau et al., 1993). Among the chemoresistance-related genes the multidrug resistance gene (mdr1) was shown to be involved in reduced response of brain tumors to chemotherapy (Matsumoto et al., 1991). Thus, the overcoming of multidrug resistance provides an attractive goal for a more effective therapy of brain tumors. Beside the occurrence of multidrug resistance (MDR) in patients with malignant gliomas, deficiencies in immunological functions have been observed which may contribute to tumor progression.
Cytokines, including tumor necrosis factor alpha (TNF.alpha.), play an important role in immunoregulation and antitumor activities. This fact led to several In Vitro and In Vivo investigations and clinical trials for an immunotherapy of malignant gliomas with TNF.alpha. (for review see: Jaeckle, 1994). Tumor necrosis factor is predominantly produced by activated macrophages and can be induced by Natural Killer-(NK), T- and tumor cells (for review see: Tracey and Cerami, 1993). Despite the fact that TNF.alpha. is an effective antitumor agent, external TNF.alpha. application in glioblastoma patients is surrounded by controversy. New approaches including gene transfer of cytokine genes into glioblastoma cells are being used in an attempt to circumvent the disadvantages of immunotherapy and to potentiate immune response against the tumor (Yu et al., 1993; Sparmann et al., 1994). Furthermore, it has been demonstrated that cytokines are able to modulate mdr1 expression and to enhance cytotoxicity to certain tumor cell lines (Evans and Baker, 1992; Walther and Stein, 1994).
The transfer of the human TNF.alpha. (hTNF) gene into U373MG human glioblastoma cells was used to investigate the effects of the cytokine on P-glycoprotein expression and the chemosensitivity to MDR related drugs in stably transfected glioblastoma cells. Introduction of the hTNF-expressing retroviral vector pN2tk-hTNF into U373MG glioblastoma cells revealed that secretion of biologically active hTNF alters growth characteristics, reduces P-glycoprotein expression associated with an enhanced rhodamine-123 uptake and accumulation, and increases cytotoxicity of doxorubicin and vincristine in vitro. This indicates that the endogenous expression of the cytokine is capable of reversing the MDR phenotype and making these cells more susceptible to treatment with MDR relevant drugs, thus providing an alternative approach in the therapy of malignant gliomas.